TY - JOUR
T1 - Membrane type-1 matrix metalloproteinase functions as a proprotein self-convertase
T2 - Expression of the latent zymogen in Pichia pastoris, autolytic activation, and the peptide sequence of the cleavage forms
AU - Rozanov, Dmitri V.
AU - Strongin, Alex Y.
PY - 2003/3/7
Y1 - 2003/3/7
N2 - An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies. A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway. To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris. Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography. Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis. This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites. Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR ↓ Y112, thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr112. The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp119 and at Asn130) and, next, to the three inactive ectodomain forms (N terminus at Thr222, at Gly284, and at Thr299). These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation. Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.
AB - An understanding of the regulatory mechanisms that control the activity of membrane type-1 matrix metalloproteinase (MT1-MMP), a key proteinase in tumor cell invasion, is essential for the design of potent and safe anti-cancer therapies. A unique proteolytic pathway regulates MT1-MMP at cancer cell surfaces. The abundance of proteolytic enzymes in cancer cells makes it difficult to identify the autocatalytic events in this pathway. To identify these events, a soluble form of MT1-MMP, lacking the C-terminal transmembrane and cytoplasmic domains, was expressed in Pichia pastoris. Following secretion, the latent zymogen and active enzyme were each purified from media by fast protein liquid chromatography. Trace amounts of active MT1-MMP induced activation of the zymogen and its self-proteolysis. This autocatalytic processing generated six main forms of MT1-MMP, each of which was subjected to the N-terminal microsequencing to identify the cleavage sites. Our data indicate that MT1-MMP functions as a self-convertase and is capable of cleaving its own prodomain at the furin cleavage motif RRKR ↓ Y112, thus autocatalytically generating the mature MT1-MMP enzyme with an N terminus starting at Tyr112. The mature enzyme undergoes further autocatalysis to the two distinct intermediates (N terminus at Trp119 and at Asn130) and, next, to the three inactive ectodomain forms (N terminus at Thr222, at Gly284, and at Thr299). These findings provide, for the first time, a structural basis for understanding the unconventional mechanisms of MT1-MMP activation and regulation. Finally, our data strongly imply that MT1-MMP is a likely substitute for the general proprotein convertase activity of furin-like proteinases, especially in furin-deficient cancer cells.
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U2 - 10.1074/jbc.M213246200
DO - 10.1074/jbc.M213246200
M3 - Article
C2 - 12514192
AN - SCOPUS:0037424402
SN - 0021-9258
VL - 278
SP - 8257
EP - 8260
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 10
ER -